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Abstract

Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

Any successful Hi-C experiment should reveal the distance-dependent decay of contact probabilities: the frequency by which two loci juxtapose in three-dimensional space is predominantly determined by their distance in the linear genome (Lieberman-Aiden et al., 2009). This observation motivated the application of Hi-C for physical mapping: the number of Hi-C links between pairs of contigs of a whole-genome shotgun assembly can serve as a proxy of the linear distance between them. This distance information can then be fed into graph algorithms to reconstruct the linear order of scaffolds along the chromosomes (Burton et al., 2013; Kaplan and Dekker, 2013). The three-dimensional proximity information obtained from our TCC approach was employed to order and orient BAC-based super scaffolds in the high-quality barley reference genome (Beier et al., 2017; Mascher et al., 2017). For the Emmer genome assembly, the TCC information was used in a similar manner to validate the scaffolds leading to chromosome-scale assemblies (pseudomolecules) (Avni et al., 2017).

TCC is a modified Hi-C approach in which key reactions (marking of the DNA ends and circularization) are performed on a solid phase rather than in solution (Kalhor et al., 2011). In fact, in human cells the tethering approach yielded improved signal-to-noise ratios, thus leading to a better mapping of low-frequency interactions (Kalhor et al., 2011). This observation prompted us to develop a similar protocol for Triticeae, which is based on the isolation of crosslinked nuclei from young leaves (Hövel et al., 2012) followed by TCC (Kalhor et al., 2011) (Figure 1). Briefly, the chromosome conformation is captured in native leaves by chemical crosslinking using formaldehyde (Hövel et al., 2012), which covalently connects proteins to DNA, as well as proteins to each other. Nuclei are purified, and cysteine residues of proteins contained in the chromatin are biotinylated (Kalhor et al., 2011). The DNA is digested with a restriction enzyme (HindIII) followed by immobilization at low density on streptavidin-coated beads via the crosslinked biotinylated proteins (Kalhor et al., 2011). DNA ends are filled-in, marked with biotin and circularized while tethered to the beads (Kalhor et al., 2011). The crosslinks are reversed, ligation junctions are affinity-purified and provided with adapters for Illumina sequencing to discover genuine pairs of intrachromosomal interaction sites, which were initially captured (Kalhor et al., 2011).

Chromosome conformation capture sequence data is analyzed in the context of genome sequence assemblies. If a chromosome-scale reference genome is available, Hi-C (or TCC) reads can be aligned to it and assigned to restriction fragment predicted in silico from the genome sequence. Subsequently, the number of Hi-C reads linking pairs of restriction pairs can be quantified and the counts be aggregated in larger genome windows (e.g., 1 Mb bins) to obtain estimates of the contact probabilities between pairs of genomic loci (Lieberman-Aiden et al., 2009). These contact probabilities can be used to investigate chromatin organization and its interaction with various biological parameters such as stage of the cell cycle (Naumova et al., 2013) or the epigenomic landscape (Zhou et al., 2013). Chromosome conformation capture sequencing can also be used to reconstruct contiguous, chromosome-scale genomic reference sequences from fragmented sequence assembly. The Hi-C/TCC reads are mapped to a sequence assembly composed of unordered scaffolds, and links between scaffolds are counted and used as a measure of genomic distance. The closer two sequence scaffolds are to each other in the linear genome, the higher the number of Hi-C read pairs linking them. This distance information can then be used to derive a linear order of sequence scaffolds and orient adjacent scaffolds relative to each other (Burton et al., 2013; Kaplan and Dekker, 2013). These approaches have been applied in the sequence assembly of insect (Dudchenko et al., 2017) and Triticeae genomes (Avni et al., 2017; Mascher et al., 2017).

Figure 1. Schematic overview of TCC for Triticeae. Leaves of seven days old plants (I) are harvested for chemical crosslinking of the chromatin (II). The chromatin structure is captured by the formation of covalent bonds between proteins (grey ovals) and between proteins and DNA (line). The orange and light blue segments exemplify two HindIII fragments located on a chromosome. Nuclei are purified (III), and proteins are biotinylated (IV, green pentagon) for subsequent tethering of the complexes to a solid phase. DNA is digested (scissors) using the restriction enzyme HindIII (V) and tethered at low density to streptavidin-coated beads (VI, purple). Ends are marked and filled-in using biotin-14-dCTP (VII, red star). By including dGTPαS in the fill-in reaction, a phosphorothioate bond is introduced. Thereby DNA is guarded against Exonuclease III digestion, which is used at a later step to remove biotinylated nucleotides from non-ligated ends (not shown). Filled-in HindIII sites are ligated ‘on-bead’ (VIII), thereby newly creating NheI restriction sites, which are indicating TCC ligation events. The crosslinking is reversed. DNA is purified (IX) and treated with Exonuclease III (not shown) prior to fragmentation (X). The position of primers (grey triangles) used for controlling the marking and ligation of ends (3C control) is indicated (IX). Ends of the fragmented DNA are repaired and tailed with ‘A’ (not shown). DNA fragments with genuine ligation junctions are affinity-purified based on the incorporated biotin (XI) and provided with Illumina adapters (XII, grey lines) for paired-end sequencing. Ligation products are PCR-amplified (XII; arrowheads: position of primer), size-selected (XIII) and quality controlled (QC). The junctions are revealed by paired-end (PE) sequencing using an Illumina HiSeq2500 device (XIV) followed by bioinformatic analysis (XV). The estimated hands-on time is indicated in days (d). The operating period of the Illumina HiSeq2500 instrument is given for 2x 100 cycles sequencing and depends on the chemistry and type of flowcell (days in brackets). The flowchart is adapted from (Kalhor et al., 2011).

Materials and Reagents

Notes:

We tested all components in independent experiments. However, this list does not imply that alternative products from other manufacturers cannot perform just as well.

The use of the published equipment and chemistry is not indicating any competing interest.

Software and computer hardware:
To analyze Hi-C/TCC sequence data, a computer server running a Unix operating system (e.g., Linux, Solaris, MacOS; see Note 1) or access to a cloud-computing system (e.g., CyVerse, http://www.cyverse.org) is required. Common UNIX command line tools (such as) need to be available. To accelerate CPU-intensive steps such as read alignment, access to a multi-core machine (> 16 CPU cores) is recommended. Depending on the number of samples to be analyzed, hard disk storage space needs to be allocated.
The following bioinformatics software need to be installed to carry out the primary data analysis described below:

All procedures can be performed at room temperature unless specified otherwise.

In order to avoid contaminations of the sequencing libraries, filter tips and gloves should be used.

Surfaces should be cleaned for DNA removal with commercial products at regular intervals.

Lab space and instruments for handling samples pre- and post-PCR must be separated physically.

Although the protocol worked well (> 40 independent experiments), the authors take no liability for the success of the experiments conducted by the reader.

Plant growth and harvesting

About 100 barley seeds are planted in two pots (16 cm diameter) filled with compost soil. Water thoroughly, and grow the plants for 7 days in a greenhouse (sodium halogen lamps, light period of 16 h, night: 18 °C and day: 21 °C). As an example, growth conditions for barley are given in (Zimmermann et al., 2006). Other plant species may require different conditions. Adapt the plant cultivation accordingly. Use young tissues (unexpanded cells) with a favorable content of nuclei.

Grow sufficient plants to obtain about 2.4 g of leaves.

Harvest the leaves with scissors. Wrap the leaves in aluminum foil and store on ice for the transport to the lab until ready for next step.

Chemical crosslinking

Trim the leaves to 0.5-1.0 cm long segments and transfer about 0.8 g tissue into a 50 ml tube. Prepare 3 tubes and store on ice.

Add inside the fume hood 15 ml cold NIBF to each tube. Push a polystyrene plug down to the liquid level for keeping the leaves submersed in the buffer during vacuum infiltration (Hövel et al., 2012) (Figure 2).

Figure 2. Tube setup during vacuum infiltration. A polystyrene plug fitting snugly to the tube is pushed down to the liquid surface area in order to ensure submersion of the leaf segments during the infiltration.

Place tubes in a desiccator and infiltrate the NIBF (150 mbar). Cut the vacuum abruptly after 5, 10 and 15 min to help the entering of the NIBF into the leaf segments. Continue with the infiltration for a total time of 1 h. The green color of segments will be darker after the infiltration (Figure 3) due to the penetration of the fixative into the intercellular space (Hövel et al., 2012) (see Note 2).

Figure 3. Leaf color change. The image is taken from a typical leaf segment before (A) and after (B) vacuum infiltration with NIBF buffer (fixative).

Cut the vacuum, remove the polystyrene plugs and add 2.0 ml of 2 M glycine to stop crosslinking (Hövel et al., 2012). Mix the sample thoroughly by pipetting up and down using a disposable 25 ml plastic pipette. Insert the plugs and apply vacuum (150 mbar) for 5 min.

Dispose the plugs and decant the liquid into a waste container. Use a sieve to keep the leaf segments in the 50 ml tube (Figure 4). Wash the leaves three times by adding 40 ml purified water per tube.

Dry the segments of each tube well with paper towel and store them on ice until grinding (Figure 4).

Figure 4. Washing and drying of leaf segments after chemical crosslinking. A. Segments are washed with purified water. A sieve is used to withhold the segments in the tube during decantation. B. Leaf segments from each tube (about 0.8 g) are dried thoroughly on a paper towel to facilitate subsequent grinding in liquid nitrogen.

Cell disruption and nuclei isolation

Precool mortar and pestle in liquid nitrogen (see Note 3). Grind about 0.8 g leaves to a fine powder. Use a metal spoon (precooled in liquid nitrogen) to transfer the powder into a pre-cooled 50 ml plastic tube (Figure 5A). Close the tube with a lid, which is punctured several times to ensure pressure balance. Store the material immediately at -80 °C. Prepare the remaining 2 tubes accordingly. Process the samples for nuclei isolation within 1 week.

Place the 3 tubes containing the powder on ice after removing the tubes from the -80° freezer and add 10 ml NIBP (Hövel et al., 2012) to each tube using a 25 ml plastic pipette. Mix carefully with a pipette to resuspend frozen clumps completely.

In order to prevent filters from clogging, distribute the suspension evenly into four funnels prepared in advance (as described below).

Filter the suspension at 4 °C through Miracloth and Sefar Nitex placed in a funnel as described (Hövel et al., 2012). Miracloth should face the plant material and Sefar Nitex the funnel surface. Use gravity flow only to avoid contamination with cell debris. Collect the filtrate in a 50 ml plastic tube (Figure 5B).

Figure 5. Cell disruption and filtering of nuclei. A. Leaf segments are ground in liquid nitrogen to a fine powder. The material is transferred into a 50 ml tube using a metal spoon and immediately stored at -80 °C. B. For the isolation of nuclei, the powder is resuspended in 10 ml NIBP and filtered through Miracloth and Sefar Nitex. The extract is collected in a clean 50 ml tube for subsequent centrifugation.

Transfer the suspension to four 1.5 ml tubes and spin in a pre-cooled centrifuge (1,900 x g, 5 min, 4 °C). Discard the supernatant and resuspend the pellets in 300 µl ice-cold NIBS.

Prepare four 2.0 ml tubes each containing 1.5 ml ice-cold sucrose cushion and carefully layer 300 µl of the suspension on top essentially as described (Abdalla et al., 2009) (Figure 6). Spin the four tubes in a pre-cooled centrifuge (16,000 x g, 1 h, 4 °C). Remove the supernatant completely and avoid contaminating the colorless nuclei pellet with the pale green top layer.

Figure 6. Sucrose cushion. The pale green nuclei extract (e) is layered on top of a sucrose cushion (c) and centrifuged in a 2.0 ml tube for further purification.

Resuspend each of the four nuclei pellets in 100 µl ice-cold NIBS. Pool two nuclei suspensions (200 µl) in a 1.5 ml tube. Place the two pools on ice and perform a microscopic quality check.

Microscopic quality and quantity check of the nuclei (optional)Note: The microscopic check might be skipped, if nuclei are purified in a routine manner, or if an epifluorescence microscope is not available.

Stain another aliquot of the nuclei suspension with VECTASHIELD mounting medium with DAPI for quantification. Count the nuclei using a cell counting chamber (Neubauer chamber) and the epifluorescence microscope (20x objective, 200-fold magnification, DAPI-filter, absorption 358 nm, emission 461 nm). Determine the concentration of the nuclei and continue the TCC library construction with approximately 107 nuclei.

Incubate the two tubes horizontally in an incubator cabinet (37 °C, overnight, gentle agitation on a rocking platform).

Pool the two digestions and inject the sample (about 2.5 ml) into one pre-wetted Slide-A-LyzerTM Dialysis cassette (20 kDa, 3 ml) according to the manufacturer’s instructions. Inject slowly to avoid shearing of the DNA (Figure 8). Aspirate most of the remaining air in the cassette, fill 1 L dialysis buffer into a beaker glass, add a magnetic stir bar and dialyze the cassette inserted into a float buoy with slow agitation for 3 h, thereby removing excess IPB from the biotinylation reaction (Kalhor et al., 2011).

Figure 8. Dialysis. After the HindIII digestion, the sample (about 2.5 ml) is injected slowly into a pre-wetted Slide-A-LyzerTM Dialysis cassette. Prior to dialysis, most of the air is aspirated from the inside. The cassette is inserted into a float buoy and placed into dialysis buffer for a total of 4 h.

Discard the buffer and dialyze for 1 h in 1 L fresh dialysis buffer.

Collect the DNA as described by the manufacturer of the dialysis cassette. Draw the sample slowly into the syringe to avoid shearing.

Reclaim the beads by mounting the five 15 ml tubes horizontally on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96). Fasten the tubes securely to the magnet by using tape or a rubber strap (Figure 9).

Figure 9. Recovery of tethered circularization products. Tubes containing the ligation products are mounted on a DynaMag-96 Side Skirted Magnetic Particle Concentrator. The beads are reclaimed, and the supernatant is discarded while the tubes are placed in the magnet.

Discard the supernatant. Perform a pulse-spin and remove liquid remnants.

Dry the pellet (5 min, room temperature).

Dissolve the five DNA pellets in 30 µl EB per tube. Pool the four TCC samples in one 1.5 ml tube. Precipitate the DNA of the TCC and 3C control by adding 1/10 volume 3 M sodium acetate, pH 5.2 and two volumes ice-cold 100% ethanol.

Mix well by inversion and incubate for 30 min on ice.

Collect the DNA by centrifugation (4 °C, 13,000 x g, 30 min).

Carefully remove the supernatant from the two tubes.

Add 500 µl ice-cold 80% ethanol.

Spin the DNA (4 °C, 13,000 x g, 15 min).

Discard the supernatants. Perform a pulse-spin and remove liquid remnants.

Dry the pellets (5 min, room temperature).

Dissolve the TCC sample in 50 µl EB and the 3C control in 10 µl EB.

Determine the DNA concentration using the Qubit 2.0 (or Qubit 4) fluorometer and the dsDNA BR Assay. The concentration should be 100-800 ng/µl.

Figure 10. Typical control for marking and ligation of ends. Two close genomic barley HindIII fragments are PCR-amplified across their ligation junction. HindIII-digestion of the amplified 3C junctions, in which HindIII sticky-ends are ligated, yields two fragments of a different size. In contrast, TCC junctions are created from the blunt-end ligation of filled-in and marked HindIII ends. As a result, novel NheI sites are formed and the original HindIII sites are lost (Belton et al., 2012). Typically, approximately 50-80% of the PCR-products are cleaved by NheI. The size of the products is indicated (bp). The gel images are taken from Mascher et al. (2017).

Titration PCR: After an initial incubation at 98 °C (30 sec), perform the amplification of the DNA (20 cycles: 98 °C for 10 sec, 68 °C for 30 sec, and 72 °C for 30 sec), followed by a final 5 min extension at 72 °C.

Take 4 µl aliquots after 9, 12, 15 and 20 cycles.

Analyse the aliquots using a standard agarose gel (Figure 11). Estimate the amount of products by comparing to a known DNA quantity from the ladder. Choose the number PCR cycles and numbers of reactions (25 µl volume) to produce approximately 200-600 ng DNA for sequencing (see Note 12). In order to avoid PCR artifacts indicated by changes in size distribution of the PCR products, it is recommended to use at most 15 cycles (Belton et al., 2012). The library shown in Figure 11 yielded sufficient amounts of DNA at 12 cycles.

Figure 11. Titration PCR. After the indicated number of PCR cycles, 2.4 µl of the reaction (initial volume of PCR: 25 µl) is withdrawn, size-separated using a standard 2% agarose gel and stained with ethidium bromide. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp). The 500 bp marker band corresponds to 20 ng DNA.

Resuspend the bead-bound TCC DNA and set up the final PCR with the calculated number of reactions. Perform PCR using the optimal number of cycles.

Reaction clean-up and size selection

Pool all PCR-products in one 1.5 ml tube and place the tube in an MPC.

Wait for 1 min and transfer the supernatant containing the library into a new 1.5 ml tube for clean-up and size selection (Belton et al., 2012).

Remove the comb and place the gel in an electrophoresis chamber filled with 1x TAE buffer. Load the TCC library in two adjacent slots. Apply the standard to one lane and perform electrophoresis for 1 h with 5 V/cm (distance between anode and cathode). Protect the gel from light during separation. Visualize the DNA using a ‘Dark reader’ transilluminator (see Note 15) and take a photograph. Use a clean scalpel and excise the region between 300 and 550 bp (Figure 12).

Figure 12. Gel-purification of a typical TCC library. Following size-separation (standard 2% agarose gel electrophoresis) and staining with SYBR Gold, the library is revealed using a visible blue light emitting ‘Dark reader transilluminator’. The DNA from the framed area between approximately 300 and 550 bp is extracted for sequencing. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp).

Place the gel block in a 2.0 ml tube and determine the volume using a balance (e.g., block of 234 mg corresponds to 234 µl).

Purify the DNA using the ‘MinElute Gel Extraction Kit’ essentially as described by the manufacturer. Add 6 volumes QG buffer and dissolve the gel completely under gentle agitation.

Add one gel volume isopropanol and mix by inversion.

Apply 700 μl of the dissolved gel to a MinElute column. Place the column in a collection tube and spin (16,000 x g, 1 min). Discard the flow-through.

Figure 13. Quality control of a final TCC library. After digestion with NheI the TCC library (80 ng) is size-separated (standard 2% agarose gel), stained with ethidium bromide and compared to the uncut control (80 ng). The shift to a smaller size distribution is indicative of the presence of NheI sites, which originate from genuine TCC ligation events (Belton et al., 2012; Mascher et al., 2017). In general, we found that the shift is a secure quality indicator. Libraries without a clear shift should not be sequenced, because they will yield only a very small fraction of useful TCC reads. The size of the GeneRuler 50 bp DNA ladder (L) is indicated (bp).

Record the size profile of the library and determine the average size of the TCC library using the Agilent High Sensitivity DNA Kit with the Agilent 2100 Bioanalyzer (Figure 14).

Trim the reads at the junction site with cutadapt (Martin, 2011) using the adapter sequence 'AAGCTAGCTT'.

Align the trimmed read pairs to an appropriate reference genome with BWA mem (Li, 2013). The two reads should be mapped as single ends by specifying the parameters ‘-S’ and ‘-P’. The parameter ‘-M’ should be used to mark shorter split hits as secondary.

Assign reads to restriction fragments with BEDTools (Quinlan and Hall, 2010). You will need a BED file (https://genome.ucsc.edu/FAQ/FAQformat.html#format1) with the positions of restriction fragments (i.e., regions between two HindIII sites) obtained from a HindIII in silico digest of your reference genome sequence. Use the command ‘bedtools pairtobed -bedpe -f 1 -type both -abam’. The resulting BEDPE file is a tab-separated file that can be processed with standard UNIX text processing tools such as AWK or Perl. The assignments of the two ends of a pair to restriction fragments are on adjacent lines.

Use UNIX scripts (e.g., in the AWK language) to calculate the insert size based on the distance of alignment start positions to neighboring HindIII sites. Discard fragments with insert sizes above 500 bp, plot the insert size distribution (Figure 15) and compare it to that obtained with the Agilent Bioanalyzer.

Figure 15. Typical in silico size profile of a TCC library. The sequence data are mapped to a reference genome and assigned to restriction fragments. The insert size of each sequenced fragments is determined from the distance of alignment start positions to the next HindIII restriction site. The mode of the distribution is indicated by a red line.

Notes

Approach the local sales representative for ordering, performance and accessory items.

If darkening of the leaves is not observed, the fixative might not have entered completely, thus indicating an incomplete crosslink. Optimize the infiltration conditions for example by increasing the vacuum or cutting shorter plant segments.

It is mandatory to wear appropriate protective gear. Consult the Safety Officer of your institution for proper handling of liquid nitrogen.

The MyOne Streptavidin T1 beads provide about 250 cm2 surface per ml. By using 400 µl beads, the purified chromatin is tethered at low density on a large surface of about 100 cm2 (Kalhor et al., 2011). Thereby intramolecular ligation of DNA ends is favored.

Free streptavidin residues are saturated with biotin in order to avoid interference with biotin-14-dCTP, which is used for marking the DNA ends during the following step (Kalhor et al., 2011).

During this reaction blunt DNA ends are generated, which are labeled with a biotinylated cytosine residue located 3’ to a phosphorothioate bond introduced by the incorporation of dGTPαS (Kalhor et al., 2011).

Spin the samples in a centrifuge equilibrated to room temperature. Cold temperatures might result in the formation of a turbid water phase. Avoid contaminating the water phase with protein from the interphase during the transfer.

NaCl is used for DNA precipitation, because the solution contains SDS (Green and Sambrook, 2012). Subsequently, the DNA is precipitated in the presence of sodium acetate to remove traces of sodium chloride, which is inhibitory to Exonuclease III (Hoheisel, 1993).

Ligation of filled-in HindIII sites (AAGCTT) generates sites for the restriction enzyme NheI (GCTAGC). To control for effectiveness of fill-in and blunt-end ligation, a PCR fragment (534 bp) from two adjacent barley HindIII restriction fragments is generated. For amplification, primer 1 and primer 2 are used.

Primers 3 and 4 anneal to the Illumina adapter and allow for the amplification of the library.

In routine experiments 11-12 amplification cycles are sufficient. Eight PCR reactions (25 µl volume per reaction) should yield enough DNA for sequencing. Follow the ‘golden rule’ to sustain the complexity of libraries: Keep the number of cycles low and increase the number of PCR reactions to obtain sufficient library for sequencing (Belton et al., 2012).

The BluePippin from Sage Science is an alternative to standard gel-purifications.

Do not use ethidium bromide stained gels. Ultra-violet radiation used for excitation of ethidium bromide will damage the DNA.

DNA is revealed in agarose gels using SYBR-Gold dye and visible blue light emitted from a 'Dark reader' transilluminator as the excitation source.

Place the columns in the rotor consistently, since the columns will be turned 180° for removing traces of liquid.

Recipes

Notes:

All solutions are prepared using purified water (GenPure Pro UV/UF) and stored at room temperature unless specified otherwise.

Good laboratory practice has to be applied throughout the experiment.

It is especially important to consult the Safety Data Sheets and the laboratory/environmental safety rules of your institution for proper handling the reagents and instruments.

The 50x TAE stock is diluted with water to 1x TAE working solution and mixed thoroughly

Acknowledgments

This work was financially supported by core funding of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, and project funding (‘TRITEX’) from the German Federal Ministry of Education and Research (BMBF, FKZ 0315954) to Nils Stein. The graphic design of Karin Lipfert is gratefully acknowledged. The authors thank Lala Aliyeva-Schnorr for help with microscopic analysis of nuclei. This protocol was adapted for the most part from wet-lab procedures (Hövel et al., 2012; Lieberman-Aiden et al., 2009; Kalhor et al., 2011; Belton et al., 2012) and from bioinformatics procedures described previously (Mascher et al., 2017; Beier et al., 2017). The authors declare no competing interests or conflicts of interest.